Our studies have focused on post-translational modification of chromosomal proteins in the nuclei of Walker-256 carcinosarcoma cells as well as in the nuclei of other rat tissues. In addition to the previously described modifications of these proteins (acetylation, methylation and phosphomonoester formation), we are concentrating on a new form of phosphorylation which is an acid-labile phosphoryl linkage. We propose to describe in more detail the process by which histones 1 and 4 are phosphorylated on lysine and histidine residues respectively. Experiments to date have demonstrated that exclusively old histone 4 is phosphorylated on histidines, while new histone 4 remains free of this modification. Methods required to investigate phosphorus-nitrogen linkages in proteins are primitive, at best, and thus a good deal of our effort is directed toward improving procedures. We are currently investigating high-pressure liquid chromatographic separation of N-phosphoryl amino acids as well as chemical methods of phosphorylating histidine residues in nucleosomes and histone 4.